In protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cultivar Puma), cooling to -- 10 degrees C at a rate of 1 degrees C/min results in extensive freeze-induced dehydration (osmotic contraction), and injury is manifested as the loss of osmotic responsiveness during warming. Under these conditions, several changes were observed in the freeze-fracture morphology of the plasma membrane. These included (i) lateral phase separations in the plasma membrane, (ii) aparticulate lamellae lying next to the plasma membrane, and (iii) regions of the plasma membrane and associated lamellae in various stages of lamellar-to-hexagonalII transition. These morphological changes also were observed after equilibration in 5.37 osmolal sorbitol at 0 degrees C, which produced a similar extent of dehydration as did freezing to -- 10 degrees C. In contrast, only small areas of lateral phase separation in the plasma membrane, with no observable aparticulate lamellae or hexagonalII configurations, were observed in protoplasts supercooled to -- 10 degrees C. Therefore, freeze-induced lamellar-to-hexagonalII phase transitions in the plasma membrane are a consequence of dehydration rather than subzero temperature per se. When suspensions of protoplasts isolated from cold-acclimated leaves were frozen to -- 10 degrees C, no injury was incurred, and hexagonalII phase transitions were not observed. No hexagonalII phase was observed even at -- 35 degrees C, though acclimated protoplasts are injured at this temperature.